Wolbachia dominance influences the Culex quinquefasciatus microbiota

Microorganisms present in mosquitoes and their interactions are key factors affecting insect development. Among them, Wolbachia is closely associated with the host and affects several fitness parameters. In this study, the bacterial and fungal microbiota from two laboratory Culex quinquefasciatus isolines (wild type and tetracycline-cured) were characterized by metagenome amplicon sequencing of the ITS2 and 16S rRNA genes at different developmental stages and feeding conditions. We identified 572 bacterial and 61 fungal OTUs. Both isolines presented variable bacterial communities and different trends in the distribution of diversity among the groups. The lowest bacterial richness was detected in sugar-fed adults of the cured isoline, whereas fungal richness was highly reduced in blood-fed mosquitoes. Beta diversity analysis indicated that isolines are an important factor in the differentiation of mosquito bacterial communities. Considering composition, Penicillium was the dominant fungal genus, whereas Wolbachia dominance was inversely related to that of Enterobacteria (mainly Thorsellia and Serratia). This study provides a more complete overview of the mosquito microbiome, emphasizing specific highly abundant components that should be considered in microorganism manipulation approaches to control vector-borne diseases.

One of the most studied arthropod-associated bacteria, Wolbachia pipientis (Rickettsiales), has the ability to manipulate host reproduction.In mosquitoes, cytoplasmic incompatibility (CI) is induced, producing non-viable offspring in specific mating combinations.In general, CI allows Wolbachia to spread within insect populations through maternal transmission, effectively invading uninfected host populations and resulting in a selective reproductive advantage over uninfected females 9 .Wolbachia strains are capable of inducing variable effects on the phenotypes of different host mosquito species.In natural carriers, it has been observed to increase larval survival and longevity.In addition, reproductive fitness parameters, such as fertility, fecundity, and egg viability, can be positively or negatively affected [10][11][12] .In transinfected mosquito populations, this bacterium can reduce lifespan and alter locomotor and blood-feeding behavior 13 .Overall, vectorial competence may be affected by the regulation of the mosquito immune response, with mosquitoes becoming refractory to viral or parasitic infections 13,14 .
In this work, the microbiota of the Wolbachia carrier Culex quinquefasciatus population maintained under laboratory conditions was analyzed and compared with a Wolbachia-cured mosquito isoline treated with tetracycline, which was allowed to recover its microbiota for many generations after treatment.The effect of the presence or absence of Wolbachia on fungal and bacterial diversity in both isolines throughout the development of the mosquito life cycle was studied through metagenomic analyses.

Mosquito rearing and collection
Culex quinquefasciatus naturally infected with Wolbachia (wPipSJ strain) was originally obtained from natural breeding sites in San Juan Province, Argentina, and has been maintained under insectary conditions since 2016 (Biological Control Laboratory of the INBIOTEC-CONICET, FIBA).From the established mosquito line, wild type (wt), an antibiotic-treated isoline (-tet), was generated by exposing larvae to a tetracycline hydrochloride solution (0.1 mg/mL final concentration, Sigma, St Louis, MO; Cat.No. T33 83) during development until the pupal stage.Pupae were transferred to clean water without antibiotics and reared to adulthood.Adults were allowed to feed ad libitum with 10% sucrose containing 0.05 mg/mL tetracycline hydrochloride solution for three consecutive generations.After that, healthy insects were reared in dechlorinated water without antibiotics for three generations, and the entire process was repeated for three more generations.Finally, both isolines were reared in dechlorinated water at 24 ± 1 °C and a 12:12 h light/dark photoperiod.Larvae were fed on commercial fish food microorganisms free, and adults were provided with 20% (w/v) sucrose solution ad libitum; gravid females were also fed with mice blood.The presence of Wolbachia in both mosquito lines was checked through amplification of a wsp gene fragment using the primer combination wsp-81F and wsp-691R from gDNA, as previously described 15 .Isoline -tet has remained stable free of Wolbachia, without the addition of antibiotics, for more than 6 years, being checked monthly by PCR amplification of the wsp gene.

Mosquito DNA extraction
A pool of 20 individuals from each of four groups was used for total genomic DNA extraction: (1) third instar larvae, (2) sucrose-fed males, (3) sucrose-fed females and (4) blood-fed females.Adults were first killed by freezing, and wings and legs were removed under a stereoscope.Dissected bodies were surface disinfected for 1 min with 70% ethanol, followed by 3 to 4 washes (1 min each) with sterile phosphate-buffered saline solution (PBS).Sucrose-fed adults were processed 48 h post emergence, and blood-fed females 96 h post emergence after blood digestion.For DNA extraction, DNeasy Blood & Tissue kit (QIAGEN) lysis buffer was added to insect samples in 1.5 mL tubes, frozen in liquid nitrogen and homogenized using a mechanical cell lyser and glass beads.Homogenates were incubated with 2 mg/mL proteinase K for 3 h for protein degradation and centrifuged at 12,000 rpm for 5 min.After that, the lysate was transferred to the kit column, following DNA extraction according to the manufacturer's protocol 16 .

Sequence analysis and taxonomic assignment
Paired-end reads were quality-checked by FastQC.Reads longer than 250 bp with an expected error < 1 were clustered into operational taxonomic units (OTUs) at 97% similarity using a combination of UPARSE pipeline 19 and Mothur software, applying quality and chimera removal filters.Taxonomic assignment for fungi was performed on representative sequences from each OTU using massBLASTer and SH mappings of the UNITE database 20 .Bacterial OTUs were classified using Mothur against the SILVA database.Unclassified OTUs were taxonomically identified by BLAST searches against the GenBank standard database, nucleotide collection (nr/nt).

Bioinformatic analysis
Using the raw reads for each sample, rarefaction curves were constructed in R, and bacterial and fungal filtered reads were used to estimate alpha-diversity indices in each community using vegan package 2.5 21 .For ITS2 samples, the reads of replicates were merged prior to diversity analysis.Relative abundance and heatmaps were

Bacterial alpha diversity
Five hundred and seventy-two (572) bacterial OTUs were clustered from the 16S rRNA amplicon reads.Richness values in larval samples were similar between wt and -tet isolines.In sugar-fed adults, tetracycline-treated richness decreased by at least half in comparison to wt.In blood-fed females, a contrary tendency was determined, displaying higher richness than the -tet line.In the wt, Shannon and InvSimpson indices values were highest in the early stage and decreased in the adult samples.In particular, blood-fed females showed over fivefold lower diversity than larvae.Similarly, the -tet line also presented a differential behavior: females displayed greater diversity than larvae, especially after blood-feeding (Table 1).

Fungal alpha diversity
Sixty-one (61) different fungal OTUs resulted from the total samples, with great variation between them, from 2 OTUs in wt blood-fed females to 32 in larvae.For this line, richness decreased during development from larvae to adult stage, while -tet line larvae and sugar-fed adults did not show great differences.Remarkably, blood-fed females of both isolines showed a drastic reduction in the total mycobiota (2 and 3 OTUs) (Table 2).Shannon and InvSimpson indices computed for each sample revealed the highest diversity in -tet sugar-fed females, in contrast to its counterpart in the wt line presenting the lowest values of all samples.
Considering all mosquito developmental stages, in wt, the greatest diversity was observed in larvae and decreased in adults independent of their food source.On the other hand, blood-fed samples showed very low richness but displayed higher diversity as a consequence of a more even distribution.-tet isoline sugar-fed females had the highest diversity, while wt had the lowest diversity among all mosquito stages.

Bacterial communities composition in Culex quinquefasciatus
Bacterial communities were largely dominated by the phylum Proteobacteria, with a major relative abundance of more than 70% in all samples (Fig. 1A, Table S3).Wolbachia (Anaplasmataceae) had a high relative abundance in adults of the wt line (60.6-93.4%),although it represented 15% of the larval reads.Instead, in the -tet isoline, this OTU was absent in larvae, while in adults, it occurred in a lower proportion than in wt (5.6-17.4%).In all tetracycline-treated mosquito groups, the wsp gene was never detected by PCR amplification, while it was amplified in the wt groups (results not shown) (Fig. 1B, Table S3).
Another highly represented taxon was Thorselliaceae, with the highest values in larvae and males in wt (21.3 and 2.9%, respectively), even though this family was absent in females.In contrast, in the -tet isoline, Table 1.Indices of richness and diversity in bacterial communities per Culex quinquefasciatus group in two isolines.a L larvae, M sucrose-fed male, SF sucrose-fed females, BF blood-fed females.Tetracycline treated isoline is indicated with the suffix (-tet).www.nature.com/scientificreports/Thorselliaceae was dominant in all mosquito stages.Remarkably, Serratia (Yersiniaceae) was the only genus present in both sucrose-and blood-fed females (20%).Xanthobacteraceae was prominent in wt larvae, with an abundance greater than 20%, distributed between Ancylobacter and Xanthobacter.Another remarkable OTU corresponded to Elizabethkingia (Weeksellaceae), which was more abundant in the -tet isoline, especially for females, reaching 14.2% in blood-fed and 7.61% in sugar-fed.Several bacterial genera were found to be considerably abundant in larvae samples, whereas they occurred at low frequency or were absent in adults.

Fungal communities composition in Culex quinquefasciatus
Samples in the wt isoline were dominated by Ascomycota (Fig. 2A).In particular, Penicillium had an abundance greater than 70% in all adult samples, although in larvae, one OTU of Mortierellomycotina (phylum Mucoromycota) showed equal relative abundance (Fig. 2B).Analysis of communities composition in the -tet isoline revealed a drastic reduction in Penicillium occurrence.Despite maintaining its dominance in sugar-fed mosquitoes, it was almost absent in larvae and blood-fed females.Particularly, in blood-fed females, Basidiomycota genera such as Sterigmatomyces and Trametes were identified, while the most abundant larval OTU was assigned to the basidiomycete order Geastrales.Two OTUs of filamentous fungi were more abundant in sugar-fed adults than in larvae samples, one belonging to the order Capnodiales and the other in the genus Dydimella.Both were absent  in blood-fed females.Additionally, some genera, such as Erysiphe and Rhodocollybia, were unique to sugar-fed adults of the -tet isoline.Yeasts and yeast-like genera, such as Sterigmatomyces, Debaryomyces, Rhodotorula, Candida and Malassezia, were also identified (Fig. 2, Table S4).

Beta diversity
The principal component analysis (PCA) plot for bacterial communities displayed both isolines as separate groups, with PC1 explaining that almost 91% of the variance was due to the differences between isolines (colored groups), while PC2 was associated with variance between stages (Fig. 3A).The Bray-Curtis-based NMDS showed a similar pattern, explaining the dissimilarities in the bacteriome between both populations as distant groups (Fig. 3C).In contrast, the PCA performed for fungal communities assigned most of the variation to PC1, in which samples were close-distanced.Only the wt larval sample was highly separated from the rest.NMDS showed an overlap in points representing samples from both isolines, supporting the PCA in which there was no separation of groups (Fig. 3B, D).In both microbial sets, larval samples were separated from the adults of their own isoline, indicating the presence of distinct and abundant OTUs.In addition, blood-fed female samples tended to have different communities from most samples in pairwise comparisons (Fig. 3, Tables S5 and S6).

Discussion
In this work, bacterial and fungal microbiome from two laboratory Cx. quinquefasciatus isolines (wt and -tet) were compared throughout mosquito development, including changes in female feeding.Bacterial communities in the wt line presented higher Shannon diversity in larvae than adults, as has been demonstrated in a Wolbachia carrier Ae. albopictus population 23 .On the other hand, the -tet isoline showed a variable diversity distribution between developmental stages, detecting less diversity in larvae than in females.Meanwhile, in previous research, Cx. tarsalis naturally free of Wolbachia exhibited a similar diversity index among all stages 24 , and Wolbachia-free An. atroparvus population presented greater diversity in larvae than in females 25 .All these different examples highlight the diverse stage-specific interactions between microbes and their host.In this study, the bacterial composition at the phylum level showed a dominance of Proteobacteria in all samples.In particular, OTUs such as Thorsellia, Serratia, Aeromonas and Elizabethkingia have been identified, some of which are commonly found in mosquitoes of the Culex genus [26][27][28] .The relative abundance data showed a different pattern between the most abundant genera when comparing the -tet and wt isolines.Samples with a high abundance of Wolbachia presented a reduced load of the enterobacteria Thorsellia and Serratia, and vice versa.As reported by Hegde et al. 26 , interaction network analysis based on metagenomic data from mosquitoes indicated that some bacteria could interact with each other, producing exclusion patterns in which the colonization of the microbiota by one OTU can inhibit the presence of another; finding that Serratia and Aeromonas would be co-excluded by Wolbachia in adult Cx. quinquefasciatus and Ae.albopictus.Additionally, an exclusion effect of the enterobacterium Serratia by Cedecea was experimentally demonstrated in Cx. quinquefasciatus and gnotobiotic Ae. aegypti systems 29 .Competition for ovary colonization by the acetobacterium Asaia has also been reported in natural Wolbachia carrier mosquito species 30 .Furthermore, in Anopheles, antibiotic treatment for Asaia reduction allows the vertical transmission of Wolbachia 31 .Interestingly, in Cx. tarsalis (naturally lacking Wolbachia endosymbiont) Thorsellia was present across all developmental stages in field-collected samples 24 .In this work, Thorsellia was detected in all stages of development, showing a considerable abundance increase in the -tet isoline.This would suggest that the pattern of dominant bacteria could be explained by an exclusion effect due to competition between Wolbachia and the enterobacteria.
Additionally, the influence of blood intake on the microbiome composition was also analyzed.It has been described that in the Cx.quinquefasciatus females dominated by Wolbachia, blood ingestion produced a drastic reduction in this genus in the midgut, accompanied by an increase in enterobacteria, indicating that Wolbachia would not participate in the digestion of blood 32 .Our data showed that in the wt isoline, the Wolbachia load remained at similar values, whereas the abundance of Enterobacteriaceae increased after blood ingestion.Serratia increased twice in blood-fed females than in sugar-fed females of the untreated isoline.However, the opposite trend was observed for the -tet isoline.Gaio et al. 33 conducted in vivo experiments using antibiotic-treated Ae. aegypti and found that the microbiota actively participates in blood digestion.Furthermore, Enterobacter and Serratia showed the strongest hemolytic activity in vitro.Similarly, in the Cx.pipiens midgut, fluorescently labeled Serratia was shown to increase 700-fold after blood ingestion 34 .Elizabethkingia (Weeksellaceae) and Stenotrophomonas (Xanthomonadaceae) were increased after the blood meal in both isolines, in agreement with Telang and Skinner 32 , who observed in Cx. quinquefasciatus females, an increase in the relative abundance of Elizabethkingia after being fed pig blood.In particular, E. anophelis, previously found in the midgut of Anopheles stephensi, would play a role in blood metabolism 35 .In addition, Stenotrophomonas has been reported as an indicator species of Ae. aegypti gut microbiome after being fed chicken blood 36 .
Considering the fungal communities, the highest richness was observed in wt larvae; as we have suggested, this biota might be acquired during feeding and reduced after metamorphosis 16 .Studies on Aedes larvae have revealed that microorganism communities are more similar to the microbiota present in the breeding water than between mosquito populations from different breeding sites, suggesting that the environment is the dominant factor shaping the mycobiota present in the larvae 37,38 .
Regarding fungal composition, the genus Penicillium was dominant in the sugar-fed mosquitoes, suggesting acquisition via sucrose solution.An inverse situation was observed in the blood-fed treated females, in which not only this OTU but also all fungal reads were extremely scarce.In engorged females, changes in the physiological state of the gut induce a selective environment for the bacterial microbiota 33,39 , suggesting that gut conditions could possibly affect the fungal communities in the same way.As has been reported in Ae. triseriatus females, mycobiota abundance and composition change in the midgut as a response to blood intake 40 .
Multivariate analysis indicated that there were greater differences between both isolines for bacteria and lesser differences for fungal communities, suggesting that bacterial community structure in -tet isoline was affected by the antibiotic treatment (Fig. 3) due to the broad spectral action of tetracycline 41 .In mosquitoes 42 and other insect hosts 43 , Wolbachia infection was found to affect the levels of reactive oxygen species (ROS), which are essential players in the immune system 42,43 .Additionally, the presence of Wolbachia may influence the expression of phenoloxidase genes, antimicrobial effectors, and the load of bacteria and fungi 44 .Therefore, the effect of the antibiotic treatment in the initial mosquito generations, in addition to the reduction of Wolbachia influence in the restabilization of microbial communities, could be the cause of dominance/exclusion of certain bacterial genera.
The elimination of Wolbachia in mosquitoes using tetracycline has been identified as a successful method for its cure, possibly because of its bacteriostatic effect on bacterial populations, which would allow the reconstitution of the microbiota after suspending its application 41 .In this work, Wolbachia was detected in adults of the -tet isoline at a much lower abundance than in wt.Detection of genomic sequences by NGS has much higher sensitivity than endpoint PCR amplification 45 .Nevertheless, in previous work, CI was detected by crossing the two lines 15 .
In the case of fungi, the multivariate analysis did not produce a distinctive pattern between groups; therefore, no definitive role could be assigned to them in the differentiation between isolines.However, the high dissimilarity values of the larvae and blood-fed females of both isolines with the rest of the samples suggest that developmental stage and feeding are important factors in mycobiome differentiation.
Here, we demonstrated that richness, diversity, and microbial composition of Cx. quinquefasciatus depends on the characteristics of the initial microbiota, developmental stage, and female feeding.Furthermore, we suggest that the transmission and establishment of the found microbiota could be due to many factors, such as occupation of an ecological niche or competitive exclusion by dominant OTUs such as Wolbachia, Thorsellia, Serratia or Penicillium.Moreover, differential effects on vertical, transstage, and horizontal transmission, environmental influence, or changes in intestinal homeostasis by antibiotics or blood intake could be involved.
The role of the dominant microorganisms in the development and fitness of this mosquito should be further investigated, in order to analyze their potential for biotechnological applications aimed at managing insect populations or reducing their vectorial capacity.

Figure 1 .
Figure 1.Bacterial relative abundance in wild-type and tetracycline-treated Culex quinquefasciatus isolines.(A) Bacterial community at the phylum level.(B) Heatmap of the relative abundance distribution of the most abundant bacteria in mosquito stages.Red color indicates higher relative abundance.L larvae, M sucrose-fed male, SF sucrose-fed females, BF blood-fed females.Tetracycline treated isoline is indicated with the suffix (-tet).

Figure 2 .
Figure 2. Fungal relative abundance in wild-type and tetracycline-treated Culex quinquefasciatus isolines.(A) Fungal community at the genus level.(B) Heatmap of the relative abundance distribution of the most abundant fungi in mosquito stages.Red color indicates higher relative abundance.L larvae, M sucrose-fed male, SF sucrose-fed females, BF blood-fed females.Tetracycline treated isoline is indicated with the suffix (-tet).

Figure 3 .
Figure 3. Principal component analysis (PCA) score plots and nonmetric multidimensional scale ordination (NMDS) plots of microorganism communities.Both analyses were performed for bacterial (A, C) and fungal (B, D) communities.In PCA score plots, based on OTU raw counts, X and Y axes show principal component 1 and principal component 2, which together explain 96.07% of the total variance (PC1: 90.81% and PC2: 5.26%) in 16S rRNA samples (A) and 98.01% of the total variance (PC1: 90.22% and PC2: 7.79%) in ITS2 samples (B).The results of the tetracycline-treated samples are represented in green, and the untreated samples are shown in red.L larvae, M sucrose-fed male, SF sucrose-fed females, BF blood-fed females.

Table 2 .
Summary of fungal community richness and alpha diversity per Culex quinquefasciatus group in two isolines.
a L larvae, M sucrose-fed male, SF sucrose-fed females, BF blood-fed females.Tetracycline treated isoline is indicated with the suffix (-tet).